Routine methods used to genotype mice involve isolation of DNA from partially amputated neonate's tail, toe or ear. Inevitable drawback of such techniques is animal`s pain response and increased spending of time and funds because of obligatory DNA purification. In order to implement a noninvasive and simple protocol for mouse DNA isolation, we have improved the method based on samples collected by swabbing of the inner cheek. Combining alkaline and temperature lysis it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method on three different mouse lines showed that it is highly efficient, the volume of the PCR samples could be reduced to 25 µl and fragments up to 800 bp were successfully amplified. This protocol reduces animal discomfort, shortens the time for DNA isolation, and enables amplification of larger DNA fragments with optimal success rate, thus considerably facilitating large-scale genotyping of different mouse lines.Key words: DNA isolation, buccal swabs, genotyping, PCR, mouse Transgenic mice are an indispensable experimental model in biomedical research and their successful breeding requires a simple and reliable genotyping procedure, which is typically performed using PCR (polymerase chain reaction). Obtaining high quality genomic DNA is a critical for successful PCR amplification. Although large quantities of DNA can be isolated from a variety of samples, the routinely used methods for mouse genotyping involve partial amputation of a neonate's tail, ear or toe (Hanley et al. 1991, Ren et al. 2001, Malumbres et al. 1997. Although efficient, the procedures are invasive, mutilating and the animals regularly exhibit pain response. Moreover, additional step of DNA purification after tissue lysis represents a significant burden, especially in laboratories which need to genotype a large number of different samples. Therefore, every simplification of DNA isolation procedure is welcomed, as it follows the trend in refinement of animal handling procedures, and also saves both time and money (Council of Europe, 2006.).Buccal swabs as a source of cells for DNA isolation was used to avoid the invasiveness of previously applied procedures. After swabbing of buccal mucosa, DNA could be isolated using commercially Here we report an improved simple method (Meldgaard et al. 2004) for mouse genotyping using buccal swab samples. By modifying the alkaline lysis and boiling method it was possible to reliably isolate the DNA from mouse buccal mucosa using simple cotton sticks and the basic, easily affordable chemicals. The PCR was performed in minimal volume (25 µL) and modified in order to achieve amplification of PCR products up to 800 bp. MATERIALS AND METHODS Animals and housing Sampling of buccal epithelial cells and isolation of DNAThe sampling of the buccal mucosa required some skilfulness and routine, as the sample should contain cells of buccal mucosa and not those of tongue. The mice were firmly held in the hand with their neck skin fixed between the thumb and the...

It is becoming increasingly clear that neurological diseases are multi-factorial involving disruptions in multiple cellular systems. Thus, while each disease has its own initiating mechanisms and pathologies, certain common pathways appear to be involved in most, if not all, neurological diseases described to date. Thus, it is unlikely that modulating only a single factor will be effective at either preventing disease development or slowing disease progression. A better approach is to identify small (< 900 daltons) molecules that have multiple biological activities relevant to the maintenance of brain function. Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglial cells and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. This wide range of actions suggests that fisetin has the ability to reduce the impact of age-related neurological diseases on brain function.

Non-small cell lung cancer (NSCLC) molecular biomarker testing is obligatory for determining therapy. The aim of this study was to compare immunocytochemistry (ICC) results of NSCLC predictive biomarkers between bronchoscopic and non-bronchoscopic type of cytology samples. This study included archive records of 1109 predictive ICC results (ALK, ROS1, and PD-L1). The ICC was done on bronchoscopic, and non-bronchoscopic NSCLC samples prepared as cytological smears and cytospins, using Dako EnVisionTM FLEX detection visualization system. The ALK, ROS1, and PD-L1 distribution between bronchoscopic and non-bronchoscopic samples was analysed. The future perspective of cytology in precision medicine was reconsidered. The obtained positive results of ALK, ROS1, and PD-L1 ICC were in concordance with the previously observed range. There was no statistically significant difference in ALK, ROS1, and PD-L1 ICC distribution between the bronchoscopic and non-bronchoscopic groups of samples (p=0.730). The comparison of PD-L1 expression, and, separately PD-L1 ≥50% expression, between two groups of samples showed no statistically significant difference (p=0.236; p=0.436). Bronchoscopic and non-bronchoscopic samples prepared as cytological smears and cytospins are a suitable, but underutilized resource for ALK, ROS1, and PD-L1 biomarker analysis. The implementation of optimized predictive immunocytochemistry assays to provide rapid and reliable results for limited tumour samples is necessary.

Background: Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs).Methods: DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis.Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis.Results: Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p < .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p < .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p < .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. Conclusion:DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

There are only a few systematic reports about DNA extraction from routine diagnostic cytological specimens. An inevitable drawback of such techniques is increased spending of time and funds required for obligatory DNA purification. To implement a simple protocol for human DNA isolation from cytological specimens related to lung cancer, bronchial aspirates together with samples collected by swabbing of the inner cheek and eyelid were used. By combining alkaline and temperature lyses it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method used for amplification of sex chromatin gene fragments showed that it is highly efficient. The presented protocol preserves high-quality DNA that is suitable for PCR-based assays.